Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts

Abstract

The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.

hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress

“…ATM becomes activated in response to DNA lesions or perhaps by alterations in chromatin structure through intermolecular autophosphorylation on Ser1981 (Bakkenist and Kastan, 2003). On the other hand, ATR regulates the cellular response to ultraviolet (UV) radiationinduced damage and stalled DNA replication forks and also participates in the late response to IR (Tibbetts et al, 1999;Guo et al, 2000). Activation of ATR proceeds through its association with ATR-interacting protein (Cortez et al, 2001).…”

p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase δ1

“…Phosphorylation, stabilization, and activation of p53 are accomplished at least in part by members of the replication checkpoint, namely the PI3K-like protein kinase ATR (Ataxia telangiectasia mutated (ATM) and Rad3 related) 13,14 and its substrate checkpoint kinase 1 (Chk1), 15 which have been shown to play a major role during the elimination of cultured cells in response to DNA damage and replication blocks. [16][17][18][19][20] Studies with Xenopus egg extracts have further demonstrated that immunodepletion of Pold leads to an increase in the phosphorylation of Chk1. 5 Together, this suggests that disruption of Pold might lead to the activation of the replication checkpoint and downstream effectors like p53 via the function of ATR.…”

C-terminal domain of Chk1 regulates its subcellular location and kinase activity for DNA repair

“…An SQ/TQ domain (residues 317-383) contains putative phosphorylation sites for ATM/ATR and lies within the regulatory domain. Chk1 is phosphorylated on Ser317 and Ser345 in response to replication stress or other types of DNA damage in an ATM/ATR-dependent fashion [16][17][18][19][20]. To determine which part of the Chk1 protein is required for DNA damage-induced Chk1 nuclear localization and retention, we generated fusion proteins by attaching GFP in-frame at the C-terminus of Chk1 mutants missing different parts of the regulatory domain.…”

“…ATM phosphorylates Ser317 and Ser345 of Chk1 in response to DNA damage. There is a nuclear location sequence (NLS) in motif 260-271 of Chk1 ( Figure 1) [16][17][18][19][20][21]. In yeast and mammalian cells Chk1 is located both in the cytoplasm and nuclei before DNA damage, but it accumulates in nuclei after DNA damage, indicating a distinct change in subcellular location [22].…”